Neural progenitor cell populations

ABSTRACT

This invention provides populations of neural progenitor cells, differentiated neurons, glial cells, and astrocytes. The populations are obtained by culturing stem cell populations (such as embryonic stem cells) in a cocktail of growth conditions that initiates differentiation, and establishes the neural progenitor population. The progenitors can be further differentiated in culture into a variety of different neural phenotypes, including dopaminergic neurons. The differentiated cell populations or the neural progenitors can be generated in large quantities for use in drug screening and the treatment of neurological disorders.

REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional PatentApplications Ser. No. 60/205,600, filed May 17, 2000; and No.60/257,608, filed Dec. 22, 2000. The priority applications are herebyincorporated herein by reference in their entirety.

TECHNICAL FIELD

[0002] This invention relates generally to the field of cell biology ofembryonic cells and neural progenitor cells. More specifically, thisinvention relates to the directed differentiation of human pluripotentstem cells to form cells of the neuronal and glial lineages, usingspecial culture conditions and selection techniques.

BACKGROUND

[0003] Repairing the central nervous system is one of the frontiers thatmedical science has yet to conquer. Conditions such as Alzheimer'sdisease, Parkinson's disease, epilepsy, Huntington's disease, and strokecan have devastating consequences for those who are afflicted. Traumaticinjury to the head or the spinal chord can instantly propel someone fromthe bounds of everyday life into the ranks of the disabled.

[0004] What makes afflictions of the nervous system so difficult tomanage is the irreversibility of the damage often sustained. A centralhope for these conditions is to develop cell populations that canreconstitute the neural network, and bring the functions of the nervoussystem back in line.

[0005] For this reason, there is a great deal of evolving interest inneural progenitor cells. Up until the present time, it was generallythought that multipotent neural progenitor cells commit early in thedifferentiation pathway to either neural restricted cells or glialrestricted cells. These in turn are thought to give rise to matureneurons, or to mature astrocytes and oligodendrocytes. Multipotentneural progenitor cells in the neural crest also differentiate toneurons, smooth muscle, and Schwann cells. It is hypothesized thatvarious lineage-restricted precursor cells renew themselves and residein selected sites of the central nervous system, such as the spinalchord. Cell lineage in the developing neural tube has been reviewed inthe research literature by Kalyani et al. (Biochem. Cell Biol. 6:1051,1998).

[0006] Putative multipotent neuroepithelial cells (NEP cells) have beenidentified in the developing spinal cord. Kalyani et al. (Dev. Biol.186:202, 1997) reported NEP cells in the rat. Mujtaba et al. (Dev. Biol.214:113, 1999) reported NEP cells in the mouse. Differentiation of NEPcells is thought to result in formation of restricted precursor cellshaving characteristic surface markers.

[0007] Putative neural restricted precursors (NRP) were characterized byMayer-Proschel et al. (Neuron 19:773, 1997). These cells expresscell-surface PS-NCAM, a polysialylated isoform of the neural celladhesion molecule. They reportedly have the capacity to generate varioustypes of neurons, but do not form glial cells.

[0008] Putative glial restricted precursors (GRPs) were identified byRao et al. (Dev. Biol. 188: 48, 1997). These cells apparently have thecapacity to form glial cells but not neurons.

[0009] Ling et al. (Exp. Neurol. 149:411, 1998) isolated progenitorcells from the germinal region of rat fetal mesencephalon. The cellswere grown in EGF, and plated on poly-lysine coated plates, whereuponthey formed neurons and glia, with occasional tyrosine hydroxylasepositive (dopaminergic) cells, enhanced by including IL-1, IL-11, LIF,and GDNF in the culture medium.

[0010] Wagner et al. (Nature Biotechnol. 17:653, 1999) reported cellswith a ventral mesencephalic dopaminergic phenotype induced from animmortalized multipotent neural stem cell line. The cells weretransfected with a Nurr1 expression vector, and then cocultured with VMtype 1 astrocytes. Over 80% of the cells obtained were claimed to have aphenotype resembling endogenous dopaminergic neurons.

[0011] Mujtaba et al. (supra) reported isolation of NRP and GRP cellsfrom mouse embryonic stem (mES) cells. The NRPs were PS-NCAMimmunoreactive, underwent self-renewal in defined medium, anddifferentiated into multiple neuronal phenotypes. They apparently didnot form glial cells. The GRPs were A2B5-immunoreactive, and reportedlydifferentiated into astrocytes and oligodendrocytes, but not neurons.

[0012] A number of recent discoveries have raised expectations thatembryonic cells may become a pluripotential source for cells and tissuesuseful in human therapy. Pluripotent cells are believed to have thecapacity to differentiate into essentially all types of cells in thebody (R. A. Pedersen, Scientif. Am. 280(4): 68, 1999). Early work onembryonic stem cells was done using inbred mouse strains as a model(reviewed in Robertson, Meth. Cell Biol. 75:173,1997; and Pedersen,Reprod. Fertil. Dev. 6:543, 1994).

[0013] Compared with mouse ES cells, monkey and human pluripotent cellshave proven to be much more fragile, and do not respond to the sameculture conditions. Only recently have discoveries been made that allowprimate embryonic cells to be cultured ex vivo.

[0014] Thomson et al. (Proc. Natl. Acad. Sci. USA 92:7844, 1995) werethe first to successfully culture embryonic stem cells from primates,using rhesus monkeys and marmosets as a model. They subsequently derivedhuman embryonic stem (hES) cell lines from human blastocysts (Science282:114, 1998). Gearhart and coworkers derived human embryonic germ(hEG) cell lines from fetal gonadal tissue (Shamblott et al., Proc.Natl. Acad. Sci. USA 95:13726, 1998). Both hES and hEG cells have thelong-sought characteristics of human pluripotent stem (hPS) cells: theyare capable of ongoing proliferation in vitro without differentiating,they retain a normal karyotype, and they retain the capacity todifferentiate to produce all adult cell types.

[0015] Reubinoff et al. (Nature Biotechnol. 18:399, 2000) reportedsomatic differentiation of human blastocysts. The cells differentiatedspontaneously in culture, with no consistent pattern of structuralorganization. After culturing for 4-7 weeks to high density,multicellular aggregates formed above the plane of the monolayer.Different cells in the culture expressed a number of differentphenotypes, including expression of β-actin, desmin, and NCAM.

[0016] Spontaneous differentiation of pluripotent stem cells in cultureor in teratomas generates cell populations with a highly heterogeneousmixture of phenotypes, representing a spectrum of different celllineages. For most therapeutic purposes, it is desirable fordifferentiated cells to be relatively uniform—both in terms of thephenotypes they express, and the types of progeny they can generate.

[0017] Accordingly, there is a pressing need for technology to generatemore homogeneous differentiated cell populations from pluripotent cellsof human origin.

SUMMARY

[0018] This invention provides a system for efficient production ofprimate cells that have differentiated from pluripotent cells into cellsof the neuronal or glial lineage. Populations of cells are describedwhich contain precursors for either lineage, which provide a source forgenerating additional precursor cells, the mature cells of the centralnervous system: neurons, astrocytes, or oligodendrocytes. Certainembodiments of the invention have the ability to generate cells of bothlineages. The precursor and mature cells of this invention can be used anumber of important applications, including drug testing and therapy torestore nervous system function.

[0019] One embodiment of this invention is a cell population thatproliferates in an in vitro culture, obtained by differentiating primatepluripotent stem (pPS) cells, wherein at least about 30% of the cells inthe population are committed to form neuronal cells, glial cells, orboth. A second embodiment is a cell population that proliferates in anin vitro culture, comprising at least about 60% neural progenitor cells,wherein at least 10% of the cells can differentiate into neuronal cells,and at least 10% of the cells can differentiate into glial cells. Athird embodiment is a cell population that proliferates in an in vitroculture, comprising at least about 60% neural progenitor cells, whereinat least 10% of the cells express A2B5, and at least 10% of the cellsexpress NCAM.

[0020] Certain cell populations of the invention are obtained bydifferentiating primate pluripotent stem cells, such as human embryonicstem cells. Some are obtained by differentiating stem cells in a mediumcontaining at least two or more ligands that bind growth factorreceptors. Some are obtained by differentiating pPS cells in a mediumcontaining growth factors, sorting the differentiated cells forexpression of NCAM or A2B5, and then 0 collecting the sorted cells.Certain cell populations are enriched such that at least 70% of thecells express NCAM or A2B5.

[0021] Another embodiment of this invention is a cell populationcomprising mature neurons, astrocytes, oligodendrocytes, or anycombination thereof, obtained by further differentiating a precursorcell population of this invention. Some such populations are obtained byculturing neural precursors in a medium containing an activator of cAMP,a neurotrophic factor, or a combination of such factors. As describedbelow, neurons produced by such methods may be capable of exhibiting anaction potential, may show gated sodium and potassium channels, and mayshow calcium flux when administered with neurotransmitters or theirequivalents. Included are populations of cells containing a substantialproportion of dopaminergic neurons, detectable for example by stainingfor tyrosine hydroxylase.

[0022] Also embodied in the invention are isolated neural precursorcells, neurons, astrocytes, and oligodendrocytes, obtained by selectinga cell for the desired phenotype from one of the cell populations.

[0023] Where derived from an established line of pPS cells, the cellpopulations and isolated cells of this invention will typically have thesame genome as the line from which they are derived. This means that thechromosomal DNA will be over 90% identical between the pPS cells and theneural cells, which can be inferred if the neural cells are obtainedfrom the undifferentiated line through the course of normal mitoticdivision. Neural cells that have been treated by recombinant methods tointroduce a transgene (such as TERT) or knock out an endogenous gene arestill considered to have the same genome as the line from which they arederived, since all non-manipulated genetic elements are preserved.

[0024] A further embodiment of the invention is a method of screening acompound for neural cell toxicity or modulation, in which a culture isprepared containing the compound and a neural cell or cell population ofthis invention, and any phenotypic or metabolic change in the cell thatresults from contact with the compound is determined.

[0025] Yet another embodiment of the invention is a method for obtaininga polynucleotide comprising a nucleotide sequence contained in an mRNAmore highly expressed in neural progenitor cells or differentiatedcells, as described and exemplified further on in this disclosure. Thenucleotide sequence can in turn be used to produce recombinant orsynthetic polynucleotides, proteins, and antibodies for gene productsenriched or suppressed in neural cells. Antibodies can also be obtainedby using the cells of this invention as an immunogen or an adsorbent toidentify markers enriched or suppressed in neural cells.

[0026] A further embodiment of the invention is a method ofreconstituting or supplementing central nervous system (CNS) function inan individual, in which the individual is administered with an isolatedcell or cell population of this invention. The isolated cells and cellpopulations can be used in the preparation of a medicament for use inclinical and veterinary treatment. Medicaments comprising the cells ofthis invention can be formulated for use in such therapeuticapplications.

[0027] Other embodiments of the invention are methods for obtaining theneural precursor cells and fully differentiated cells of this invention,using the techniques outlined in this disclosure on a suitable stem cellpopulation. Included are methods for producing cell populationscontaining dopaminergic cells at a frequency of 1%, 3% or 5%—andpopulations of progenitor cells capable of generating dopaminergic cellsat this frequency—from primate embryonic stem cells. This isparticularly significant in view of the loss in dopamine neuron functionthat occurs in Parkinson's disease. The compositions, methods, andtechniques described in this disclosure hold considerable promise foruse in diagnostic, drug screening, and therapeutic applications.

[0028] These and other embodiments of the invention will be apparentfrom the description that follows.

DRAWINGS

[0029]FIG. 1 is a graph representing the growth of cells bearing neuralmarkers that were derived from human embryonic stem cells. The upperpanel shows growth of cells maintained in the presence of CNTF, bFGF,and NT3, and then sorted for expression of NCAM. The lower panel showsgrowth of cells maintained in the presence of EGF, bFGF, PDGF, andIGF-1, and then sorted for expression of A2B5. Four different hES celllines were used: H1, H7, H9, and H13. The A2B5 selected population hasbeen passaged over 7 times, and can be further differentiated into bothneuronal and glial cells.

[0030]FIG. 2 is a schematic diagram outlining an exemplary procedure forobtaining A2B5-positive cells. Abbreviations used: MEF-CM=mediumconditioned by culturing with mouse embryonic fibroblasts; +/−SHH=withor without sonic hedgehog; D/F12=DMEM/F12 medium; N2 and B27, culturesupplements (Gibco); EPFI=differentiation agents EGF, PDGF, bFGF, andIGF-1; PLL=poly-L lysine substrate; PLL/FN=substrate of poly-L lysineand fibronectin.

[0031]FIG. 3 is a half-tone reproduction of a fluorescence micrograph ofthe brains from neonatal rats administered with cells that express greenfluorescent protein. Left panels: parental hES cell line. Middle panels:embryoid body cells formed from the parental line. Right panels:differentiated cells sorted for expression of NCAM. Undifferentiated hEScells and embryoid body cells remain in the area of administration andshow evidence of necrosis. In contrast, the differentiated NCAM⁺cellsappear as single cells, and have migrated away from the injection site.

[0032]FIG. 4 is a photocopy reproduction of a fluorescence micrographshowing a cell staining for tyrosine hydroxylase (TH), a marker fordopaminergic cells. Embryoid bodies made from human ES cells weremaintained in 10 μm retinoic acid for 4 days, plated into aneural-supportive cocktail, and then passaged into medium containing 10ng/mL NT-3 and 10 ng/mL BDNF. Certain populations of this inventioncontain >1% TH-positive cells.

[0033]FIG. 5 is a series of graphs showing response of theneural-restricted precursors to various neurotransmitters. Panel A showsthe ratio of emission data from single cells on two differentcoverslips. Both cells responded to GABA, elevated potassium,acetylcholine and ATP. Panel B shows the frequency of cells tested thatresponded to specific neurotransmitters. Panel C shows the combinationsof neurotransmitter responses observed.

[0034]FIG. 6 is a series of graphs showing electrophysiology ofneural-restricted precursors. Panel A shows sodium and potassiumcurrents observed in two cells depolarized to test potentials between−80 and 80 mV from a holding potential of −100 mV. Panel B shows theinward (Na⁺) and outward (K) peak current-voltage relationshipsobserved. Panel C shows action potentials generated by the same cells nresponse to depolarizing stimuli. These measurements show that neuralprecursor cells derived from human ES cells are capable of generatingaction potentials characteristic of neurotransmission.

DETAILED DESCRIPTION

[0035] This invention provides a system for preparing and characterizingneural progenitor cells, suitable for use for therapeutic administrationand drug screening.

[0036] It has been discovered that when pluripotent stem cells arecultured in the presence of selected differentiating agents, apopulation of cells is derived that has a remarkably high proportion ofcells with phenotypic characteristics of neural cells. Optionally, theproportion of neural cells can be enhanced by sorting differentiatedcells according to cell-surface markers. Since certain types ofpluripotent stem cells (such as embryonic stem cells) can proliferate inculture for a year or more, the invention described in this disclosureprovides an almost limitless supply of neural precursors. Certain cellpopulations of this invention are capable of generating cells of theneuronal or glial lineage, and themselves can be replicated through alarge number of passages in culture.

[0037]FIG. 1 shows the growth curve of cells that have been culturedwith differentiating agents, and then selected according to whether theybear polysialylated NCAM, or the A2B5 epitope. Either of these cellpopulations can be proliferated through a large number of celldoublings.

[0038] Differentiated cells positively selected for A2B5 expressioncomprise cells that appear to express A2B5 without NCAM, and cells thatexpress A2B5 and NCAM simultaneously. In one of the experimentsdescribed below, maturation of these cells produced 13%oligodendrocytes, and 38% neurons. Since these cells proliferate inlong-term culture without losing their phenotype, the population canprovide a reserve of multipotential cells. Upon administration to asubject with CNS dysfunction, the population would comprise cells thatmay repopulate both the neuronal and glial cell lineage, as needed.

[0039] If desired, the neural precursor cells can be furtherdifferentiated ex vivo, either by culturing with a maturation factor,such as a neurotrophic factor, or by withdrawing one or more factorsthat sustain precursor cell renewal. Neurons, astrocytes, andoligodendrocytes are mature differentiated cells of the neural lineagethat can be obtained by culturing the precursor cells in this fashion.The neurons obtained by these methods have extended processescharacteristic of this cell type, show staining for neuron-specificmarkers like neurofilament and MAP-2, and show evidence of synapseformation, as detected by staining for synaptophysin. FIG. 5 shows thatthese cells respond to a variety of neurotransmitter substances. FIG. 6shows that these cells are capable of action potentials as measured in astandard patch-clamp system. In all these respects, the cells areapparently capable of full neurological function.

[0040] Of particular interest is the ability of this system to generatea supply of dopaminergic neurons (FIG. 4). Cells of this type areparticularly desirable for the treatment of Parkinson's disease, forwhich the best current modality is an allograft of fetal brain tissue.The use of fetal tissue as a clinical therapy is fraught with supply andprocedural issues, but no other source described previously can supplythe right kind of cells with sufficient abundance. The neural precursorcells of this invention are capable of generating differentiated cellsin which several percent of the neurons have a dopaminergic phenotype.This is believed to be a sufficient proportion for cell replacementtherapy in Parkinson's disease, and warrants the development of theprogenitor populations of this invention for therapeutic use.

[0041] Since pluripotent stem cells and some of the lineage-restrictedprecursors of this invention proliferate extensively in culture, thesystem described in this disclosure provides an unbounded supply ofneuronal and glial cells for use in research, pharmaceuticaldevelopment, and the therapeutic management of CNS abnormalities. Thepreparation and utilization of the cells of this invention isillustrated further in the description that follows.

[0042] Definitions

[0043] For the purposes of this disclosure, the terms “neural progenitorcell” or “neural precursor cell” mean a cell that can generate progenythat are either neuronal cells (such as neuronal precursors or matureneurons) or glial cells (such as glial precursors, mature astrocytes, ormature oligodendrocytes). Typically, the cells express some of thephenotypic markers that are characteristic of the neural lineage.Typically, they do not produce progeny of other embryonic germ layerswhen cultured by themselves in vitro, unless dedifferentiated orreprogrammed in some fashion.

[0044] A “neuronal progenitor cell” or “neuronal precursor cell” is acell that can generate progeny that are mature neurons. These cells mayor may not also have the capability to generate glial cells.

[0045] A “glial progenitor cell” or “glial precursor cell” is a cellthat can generate progeny that are mature astrocytes or matureoligodendrocytes. These cells may or may not also have the capability togenerate neuronal cells.

[0046] A “multipotent neural progenitor cell population” is a cellpopulation that has the capability to generate both progeny that areneuronal cells (such as neuronal precursors or mature neurons), andprogeny that are glial cells (such as glial precursors, matureastrocytes, or mature oligodendrocytes), and sometimes other types ofcells. The term does not require that individual cells within thepopulation have the capability of forming both types of progeny,although individual cells that are multipotent neural progenitors may bepresent.

[0047] In the context of cell ontogeny, the adjective “differentiated”is a relative term. A “differentiated cell” is a cell that hasprogressed further down the developmental pathway than the cell it isbeing compared with. Thus, pluripotent embryonic stem cells candifferentiate to lineage-restricted precursor cells, such ashematopoetic cells, which are pluripotent for blood cell types;hepatocyte progenitors, which are pluripotent for hepatocytes; andvarious types of neural progenitors listed above. These in turn can bedifferentiated further to other types of precursor cells further downthe pathway, or to an end-stage differentiated cell, which plays acharacteristic role in a certain tissue type, and may or may not retainthe capacity to proliferate further. Neurons, astrocytes, andoligodendrocytes are all examples of terminally differentiated cells.

[0048] A “differentiation agent”, as used in this disclosure, refers toone of a collection of compounds that are used in culture systems ofthis invention to produce differentiated cells of the neural lineage(including precursor cells and terminally differentiated cells). Nolimitation is intended as to the mode of action of the compound. Forexample, the agent may assist the differentiation process by inducing orassisting a change in phenotype, promoting growth of cells with aparticular phenotype or retarding the growth of others, or acting inconcert with other agents through unknown mechanisms.

[0049] Unless explicitly indicated otherwise, the techniques of thisinvention can be brought to bear without restriction on any type ofprogenitor cell capable of differentiating into neuronal or glial cells.

[0050] Prototype “primate Pluripotent Stem cells” (pPS cells) arepluripotent cells derived from pre-embryonic, embryonic, or fetal tissueat any time after fertilization, and have the characteristic of beingcapable under appropriate conditions of producing progeny of severaldifferent cell types that are derivatives of all of the three germinallayers (endoderm, mesoderm, and ectoderm), according to a standardart-accepted test, such as the ability to form a teratoma in 8-12 weekold SCID mice.

[0051] Included in the definition of pPS cells are embryonic cells ofvarious types, exemplified by human embryonic stem (hES) cells,described by Thomson et al. (Science 282:1145, 1998); embryonic stemcells from other primates, such as Rhesus stem cells (Thomson et al.,Proc. Nati. Acad. Sci. USA 92:7844, 1995), marmoset stem cells (Thomsonet al., Biol. Reprod. 55:254, 1996) and human embryonic germ (hEG) cells(Shamblott et al., Proc. Natl. Acad. Sci. USA 95:13726, 1998). Othertypes of pluripotent cells are also included in the term. Any cells ofprimate origin that are capable of producing progeny that arederivatives of all three germinal layers are included, regardless ofwhether they were derived from embryonic tissue, fetal tissue, or othersources. The pPS cells are not derived from a malignant source. It isdesirable (but not always necessary) that the cells be karyotypicallynormal.

[0052] pPS cell cultures are described as “undifferentiated” when asubstantial proportion of stem cells and their derivatives in thepopulation display morphological characteristics of undifferentiatedcells, clearly distinguishing them from differentiated cells of embryoor adult origin. Undifferentiated pPS cells are easily recognized bythose skilled in the art, and typically appear in the two dimensions ofa microscopic view in colonies of cells with high nuclear/cytoplasmicratios and prominent nucleoli. It is understood that colonies ofundifferentiated cells within the population will often be surrounded byneighboring cells that are differentiated. “Feeder cells” or “feeders”are terms used to describe cells of one type that are co-cultured withcells of another type, to provide an environment in which the cells ofthe second type can grow. For example, certain types of pPS cells can besupported by primary mouse embryonic fibroblasts, immortalized mouseembryonic fibroblasts, or human fibroblast-like cells differentiatedfrom hES cell. pPS cell populations are said to be “essentially free” offeeder cells if the cells have been grown through at least one roundafter splitting in which fresh feeder cells are not added to support thegrowth of the pPS.

[0053] The term “embryoid bodies” is a term of art synonymous with“aggregate bodies”. The terms refer to aggregates of differentiated andundifferentiated cells that appear when pPS cells overgrow in monolayercultures, or are maintained in suspension cultures. Embryoid bodies area mixture of different cell types, typically from several germ layers,distinguishable by morphological criteria and cell markers detectable byimmunocytochemistry.

[0054] A “growth environment” is an environment in which cells ofinterest will proliferate, differentiate, or mature in vitro. Featuresof the environment include the medium in which the cells are cultured,any growth factors or differentiation-inducing factors that may bepresent, and a supporting structure (such as a substrate on a solidsurface) if present.

[0055] A cell is said to be “genetically altered”, “transfected”, or“genetically transformed” when a polynucleotide has been transferredinto the cell by any suitable means of artificial manipulation, or wherethe cell is a progeny of the originally altered cell that has inheritedthe polynucleotide. The polynucleotide will often comprise atranscribable sequence encoding a protein of interest, which enables thecell to express the protein at an elevated level. The genetic alterationis said to be “inheritable” if progeny of the altered cell have the samealteration.

[0056] The term “antibody” as used in this disclosure refers to bothpolyclonal and monoclonal antibody. The ambit of the term deliberatelyencompasses not only intact immunoglobulin molecules, but also suchfragments and derivatives of immunoglobulin molecules (such as singlechain Fv constructs, diabodies, and fusion constructs) as may beprepared by techniques known in the art, and retaining a desiredantibody binding specificity.

[0057] General Techniques

[0058] For further elaboration of general techniques useful in thepractice of this invention, the practitioner can refer to standardtextbooks and reviews in cell biology, tissue culture, and embryology.Included are Teratocarcinomas and embryonic stem cells: A practicalapproach (E. J. Robertson, ed., IRL Press Ltd. 1987); Guide toTechniques in Mouse Development (P. M. Wasserman et al. eds., AcademicPress 1993); Embryonic Stem Cell Differentiation in Vitro (M. V. Wiles,Meth. Enzymol. 225:900, 1993); Properties and uses of Embryonic StemCells: Prospects for Application to Human Biology and Gene Therapy (P.D. Rathjen et al., Reprod. Fertil. Dev. 10:31, 1998).

[0059] For elaboration of nervous system abnormalities, and thecharacterization of various types of nerve cells, markers, and relatedsoluble factors, the reader is referred to CNS Regeneration: BasicScience and Clinical Advances, M. H. Tuszynski & J. H. Kordower, eds.,Academic Press, 1999.

[0060] Methods in molecular genetics and genetic engineering aredescribed in Molecular Cloning: A Laboratory Manual, 2nd Ed. (Sambrooket al., 1989); Oligonucleotide Synthesis (M. J. Gait, ed., 1984); AnimalCell Culture (R. I. Freshney, ed., 1987); the series Methods inEnzymology (Academic Press); Gene Transfer Vectors for Mammalian Cells(J. M. Miller & M. P. Calos, eds., 1987); Current Protocols in MolecularBiology and Short Protocols in Molecular Biology, 3rd Edition (F. M.Ausubel et al., eds., 1987 & 1995); and Recombinant DNA Methodology II(R. Wu ed., Academic Press 1995). Reagents, cloning vectors, and kitsfor genetic manipulation referred to in this disclosure are availablefrom commercial vendors such as BioRad, Stratagene, Invitrogen, andClonTech.

[0061] General techniques used in raising, purifying and modifyingantibodies, and the design and execution of immunoassays includingimmunohistochemistry, the reader is referred to Handbook of ExperimentalImmunology (D. M. Weir & C. C. Blackwell, eds.); Current Protocols inImmunology (J. E. Coligan et al., eds., 1991); and R. Masseyeff, W. H.Albert, and N. A. Staines, eds., Methods of Immunological Analysis(Weinheim: VCH Verlags GmbH, 1993).

[0062] Sources of Stem Cells

[0063] This invention can be practiced using stem cells of varioustypes, which may include the following non- limiting examples.

[0064] U.S. Pat. No. 5,851,832 reports multipotent neural stem cellsobtained from brain tissue. U.S. Pat. No. 5,766,948 reports producingneuroblasts from newborn cerebral hemispheres. U.S. Pat. Nos. 5,654,183and 5,849,553 report the use of mammalian neural crest stem cells. U.S.Pat. No. 6,040,180 reports in vitro generation of differentiated neuronsfrom cultures of mammalian multipotential CNS stem cells. WO 98/50526and WO 99/01159 report generation and isolation of neuroepithelial stemcells, oligodendrocyte-astrocyte precursors, and lineage-restrictedneuronal precursors. U.S. Pat. No. 5,968,829 reports neural stem cellsobtained from embryonic forebrain and cultured with a medium comprisingglucose, transferrin, insulin, selenium, progesterone, and several othergrowth factors.

[0065] Except where otherwise required, the invention can be practicedusing stem cells of any vertebrate species. Included are stem cells fromhumans; as well as non-human primates, domestic animals, livestock, andother non-human mammals.

[0066] Amongst the stem cells suitable for use in this invention areprimate pluripotent stem (pPS) cells derived from tissue formed aftergestation, such as a blastocyst, or fetal or embryonic tissue taken anytime during gestation. Non-limiting examples are primary cultures orestablished lines of embryonic stem cells or embryonic germ cells.

[0067] Embryonic Stem Cells

[0068] Embryonic stem cells can be isolated from blastocysts of membersof the primate species (Thomson et al., Proc. Natl. Acad. Sci. USA92:7844, 1995). Human embryonic stem (hES) cells can be prepared fromhuman blastocyst cells using the techniques described by Thomson et al.(U.S. Pat. No. 5,843,780; Science 282:1145, 1998; Curr. Top. Dev. Biol.38:133 ff., 1998) and Reubinoff et al, Nature Biotech. 18:399,2000.

[0069] Briefly, human blastocysts are obtained from human in vivopreimplantation embryos. Alternatively, in vitro fertilized (IVF)embryos can be used, or one-cell human embryos can be expanded to theblastocyst stage (Bongso et al., Hum Reprod 4: 706,1989). Embryos arecultured to the blastocyst stage in G1.2 and G2.2 medium (Gardner etal., Fertil. Steril. 69:84, 1998). The zona pellucida is removed fromdeveloped blastocysts by brief exposure to pronase (Sigma). The innercell masses are isolated by immunosurgery, in which blastocysts areexposed to a 1:50 dilution of rabbit anti-human spleen cell antiserumfor 30 min, then washed for 5 min three times in DMEM, and exposed to a1:5 dilution of Guinea pig complement (Gibco) for 3 min (Solter et al.,Proc. Natl. Acad. Sci. USA 72:5099, 1975). After two further washes inDMEM, lysed trophectoderm cells are removed from the intact inner cellmass (ICM) by gentle pipetting, and the ICM plated on mEF feeder layers.

[0070] After 9 to 15 days, inner cell mass-derived outgrowths aredissociated into clumps, either by exposure to calcium andmagnesium-free phosphate-buffered saline (PBS) with 1 mM EDTA, byexposure to dispase or trypsin, or by mechanical dissociation with amicropipette; and then replated on mEF in fresh medium. Growing colonieshaving undifferentiated morphology are individually selected bymicropipette, mechanically dissociated into clumps, and replated.ES-like morphology is characterized as compact colonies with apparentlyhigh nucleus to cytoplasm ratio and prominent nucleoli. Resulting EScells are then routinely split every 1-2 weeks by brief trypsinization,exposure to Dulbecco's PBS (containing 2 mM EDTA), exposure to type IVcollagenase (˜200 U/mL; Gibco) or by selection of individual colonies bymicropipette. Clump sizes of about 50 to 100 cells are optimal.

[0071] Embryonic Germ Cells

[0072] Human Embryonic Germ (hEG) cells can be prepared from primordialgerm cells present in human fetal material taken about 8-11 weeks afterthe last menstrual period. Suitable preparation methods are described inShamblott et al., Proc. Natl. Acad. Sci. USA 95:13726,1998 and U.S. Pat.No. 6,090,622.

[0073] Briefly, genital ridges are rinsed with isotonic buffer, thenplaced into 0.1 mL 0.05% trypsin/0.53 mM sodium EDTA solution (BRL) andcut into <1 mm³ chunks. The tissue is then pipetted through a 100 μL tipto further disaggregate the cells. It is incubated at 37° C. for ˜5 min,then ˜3.5 mL EG growth medium is added. EG growth medium is DMEM, 4500mg/L D-glucose, 2200 mg/L mM NaHCO₃; 15% ES qualified fetal calf serum(BRL); 2 mM glutamine (BRL); 1 mM sodium pyruvate (BRL); 1000-2000 U/mLhuman recombinant leukemia inhibitory factor (LIF, Genzyme); 1-2 ng/mlhuman recombinant bFGF (Genzyme); and 10 μM forskolin (in 10% DMSO). Inan alternative approach, EG cells are isolated usinghyaluronidase/collagenase/DNAse. Gonadal anlagen or genital ridges withmesenteries are dissected from fetal material, the genital ridges arerinsed in PBS, then placed in 0.1 ml HCD digestion solution (0.01%hyaluronidase type V, 0.002% DNAse I, 0.1% collagenase type IV, all fromSigma prepared in EG growth medium). Tissue is minced, incubated 11 h orovernight at 37° C., resuspended in 1-3 mL of EG growth medium, andplated onto a feeder layer.

[0074] Ninety-six well tissue culture plates are prepared with asub-confluent layer of feeder cells (e.g., STO cells, ATCC No. CRL 1503)cultured for 3 days in modified EG growth medium free of LIF, bFGF orforskolin, inactivated with 5000 rad γ-irradiation. ˜0.2 mL of primarygerm cell (PGC) suspension is added to each of the wells. The firstpassage is done after 7-10 days in EG growth medium, transferring eachwell to one well of a 24-well culture dish previously prepared withirradiated STO mouse fibroblasts. The cells are cultured with dailyreplacement of medium until cell morphology consistent with EG cells isobserved, typically after 7-30 days or 1-4 passages.

[0075] Propagation of pPS Cells in an Undifferentiated State

[0076] pPS cells can be propagated continuously in culture, usingculture conditions that promote proliferation without promotingdifferentiation. Exemplary serum-containing ES medium is made with 80%DMEM (such as Knock-Out DMEM, Gibco), 20% of either defined fetal bovineserum (FBS, Hyclone) or serum replacement (WO 98/30679), 1%non-essential amino acids, 1 mM L-glutamine, and 0.1 mMγ-mercaptoethanol. Just before use, human bFGF is added to 4 ng/mL (WO99/20741, Geron Corp.).

[0077] Traditionally, ES cells are cultured on a layer of feeder cells,typically fibroblasts derived from embryonic or fetal tissue. Embryosare harvested from a CF1 mouse at 13 days of pregnancy, transferred to 2mL trypsin/EDTA, finely minced, and incubated 5 min at 37° C. 10% FBS isadded, debris is allowed to settle, and the cells are propagated in 90%DMEM, 10% FBS, and 2 mM glutamine. To prepare a feeder cell layer, cellsare irradiated to inhibit proliferation but permit synthesis of factorsthat support ES cells (˜4000 rads γ-irradiation). Culture plates arecoated with 0.5% gelatin overnight, plated with 375,000 irradiated mEFsper well, and used 5 h to 4 days after plating. The medium is replacedwith fresh hES medium just before seeding pPS cells.

[0078] Scientists at Geron have discovered that pPS cells canalternatively be maintained in an undifferentiated state even withoutfeeder cells. The environment for feeder-free cultures includes asuitable culture substrate, particularly an extracellular matrix such asMatrigel® or laminin. The pPS cells are plated at >15,000 cells cm⁻²(optimally 90,000 cm⁻² to 170,000 cm⁻²). Typically, enzymatic digestionis halted before cells become completely dispersed (say, ˜5 min withcollagenase IV). Clumps of ˜10-2000 cells are then plated directly ontothe substrate without further dispersal.

[0079] Feeder-free cultures are supported by a nutrient medium typicallyconditioned by culturing irradiated primary mouse embryonic fibroblasts,telomerized mouse fibroblasts, or fibroblast-like cells derived from pPScells. Medium can be conditioned by plating the feeders at a density of˜5.6×10⁴ cm⁻² in a serum free medium such as KO DMEM supplemented with20% serum replacement and 4 ng/mL bFGF. Medium that has been conditionedfor 1-2 days is supplemented with further bFGF, and used to support pPScell culture for 1-2 days.

[0080] Under the microscope, ES cells appear with highnuclear/cytoplasmic ratios, prominent nucleoli, and compact colonyformation with poorly discernable cell junctions. Primate ES cellsexpress stage-specific embryonic antigens (SSEA) 3 and 4, and markersdetectable using antibodies designated Tra-1-60 and Tra-1-81 (Thomson etal., Science 282:1145, 1998). Mouse ES cells can be used as a positivecontrol for SSEA-1, and as a negative control for SSEA-4, Tra-1-60, andTra-1-81. SSEA-4 is consistently present on human embryonal carcinoma(hEC) cells. Differentiation of pPS cells in vitro results in the lossof SSEA-4, Tra-1-60, and Tra-1-81 expression and increased expression ofSSEA-1. SSEA-1 is also found on hEG cells.

[0081] Materials and Procedures for Preparing Neural Precursors andTerminally Differentiated Cells

[0082] Certain neural precursor cells of this invention are obtained byculturing, differentiating, or reprogramming stem cells in a specialgrowth environment that enriches for cells with the desired phenotype(either by outgrowth of the desired cells, or by inhibition or killingof other cell types). These methods are applicable to many types of stemcells, including primate pluripotent stem (pPS) cells described in theprevious section.

[0083] Typically, the differentiation takes place in a cultureenvironment comprising a suitable substrate, and a nutrient medium towhich the differentiation agents are added. Suitable substrates includesolid surfaces coated with a positive charge, such as a basic aminoacid, exemplified by poly-L-lysine and polyornithine. Substrates can becoated with extracellular matrix components, exemplified by fibronectin.Other permissive extracellular matrixes include Matrigel® (extracellularmatrix from Engelbreth-Holm-Swarm tumor cells) and laminin. Alsosuitable are combination substrates, such as poly-L-lysine combined withfibronectin, laminin, or both.

[0084] Suitable differentiation agents include growth factors of variouskinds, such as epidermal growth factor (EGF), transforming growth factora (TGF-α), any type of fibroblast growth factor (exemplified by FGF-4,FGF-8, and basic fibroblast growth factor=bFGF), platelet-derived growthfactor (PDGF), insulin-like growth factor (IGF-1 and others), highconcentrations of insulin, sonic hedgehog, members of the neurotrophinfamily (such as nerve growth factor=NGF, neurotrophin 3=NT-3,brain-derived neurotrophic factor=BDNF), bone morphogenic proteins(especially BMP-2 & BMP-4), retinoic acid (RA) and ligands to receptorsthat complex with gp 30 (such as LIF, CNTF, and IL-6). Also suitable arealternative ligands and antibodies that bind to the respectivecell-surface receptors for the aforementioned factors. Typically, aplurality of differentiation agents is used, which may comprise 2, 3, 4,or more of the agents listed above or in the examples below. Exemplaryis a cocktail containing EGF, bFGF, PDGF, and IGF-1 (Examples 1 and 2).

[0085] The factors are supplied to the cells in a nutrient medium, whichis any medium that supports the proliferation or survival of the desiredcell type. It is often desirable to use a defined medium that suppliesnutrients as free amino acids rather than serum. It is also beneficialto supplement the medium with additives developed for sustained culturesof neural cells. Exemplary are N2 and B27 additives, availablecommercially from Gibco.

[0086] Where the stem cells are pPS cells, the cells (obtained fromfeeder cell supported or feeder-free cultures) are differentiated byculturing in the presence of a suitable cocktail of differentiationagents.

[0087] In one method of affecting differentiation, pPS cells are plateddirectly onto a suitable substrate, such as an adherent glass or plasticsurface, such as coverslips coated with a polyamine. The cells are thencultured in a suitable nutrient medium that is adapted to promotedifferentiation towards the desired cell lineage. This is referred to asthe “direct differentiation” method.

[0088] In another method, pPS cells are first let differentiate into aheterogeneous cell population. In an exemplary variation, embryoidbodies are formed from the pPS cells by culturing them in suspension.Optionally, one or more of the differentiation agents listed earlier(such as retinoic acid) can be included in the medium to promotedifferentiation within the embryoid body. After the embryoid bodies havereached sufficient size (typically 3-4 days), they are plated onto thesubstrate of the differentiation culture. The embryoid bodies can beplated directly onto the substrate without dispersing the cells. Thisallows neural cell precursors to migrate out of the embryoid bodies andon to the extracellular matrix. Subsequent passaging of these culturesinto an appropriate medium helps select out the neural progenitor cells.

[0089] Cells prepared according to these procedures have been found tobe capable of further proliferation (Example 1). As many as 30%, 50%,75% or more of the cells express either polysialylated NCAM or the A2B5epitope, or both. Typically, at least about 10%, 20%, 30% or 50% of thecells express NCAM, and at least about 10%, 20%, 30% or 50% of the cellsexpress A2B5—which implies that they have the capacity to form cells ofthe neuronal lineage, and the glial lineage, respectively.

[0090] Optionally, the differentiated cells can be sorted based onphenotypic features to enrich for certain populations. Typically, thiswill involve contacting each cell with an antibody or ligand that bindsto a marker characteristic of neural cells, followed by separation ofthe specifically recognized cells from other cells in the population.One method is immunopanning, in which specific antibody is coupled to asolid surface. The cells are contacted with the surface, and cells notexpressing the marker are washed away. The bound cells are thenrecovered by more vigorous elution. Variations of this are affinitychromatography and antibody-mediated magnetic cell sorting. In a typicalsorting procedure, the cells are contacted with a specific primaryantibody, and then captured with a secondary anti-immunoglobulin reagentbound to a magnetic bead. The adherent cells are then recovered bycollecting the beads in a magnetic field.

[0091] Another method is fluorescence-activated cell sorting, in whichcells expressing the marker are labeled with a specific antibody,typically by way of a fluorescently labeled secondaryanti-immunoglobulin. The cells are then separated individually accordingto the amount of bound label using a suitable sorting device. Any ofthese methods permit recovery of a positively selected population ofcells that bear the marker of interest, and a negatively selectedpopulation of cells that not bear the marker in sufficient density oraccessibility to be positively selected. Negative selection can also beeffected by incubating the cells successively with a specific antibody,and a preparation of complement that will lyse cells to which theantibody has bound. Sorting of the differentiated cell population canoccur at any time, but it has generally been found that sorting is besteffected shortly after initiating the differentiation process.

[0092] It has been found that cells selected positively forpolysialylated NCAM can provide a population that is 60%, 70%, 80%, oreven 90% NCAM positive (Example 1). This implies that they are capableof forming some type of neural cell, including neurons.

[0093] It has also been found that cells selected positively for A2B5can provide a population that is 60%, 70%, 80%, or even 90% A2B5positive (Example 2). This implies that they are capable of forming sometype of neural cell, possibly including both neurons and glial cells.The A2B5 positive cells can be sorted again into two separatepopulations: one that is A2B5 positive and NCAM negative, and one thatis both A2B5 positive and NCAM positive.

[0094] Differentiated or separated cells prepared according to thisprocedure can be maintained or proliferated further in any suitableculture medium. Typically, the medium will contain most of theingredients used initially to differentiate the cells.

[0095] If desired, neural precursor cells prepared according to theseprocedures can be further differentiated to mature neurons, astrocytes,or oligodendrocytes. This can be effected by culturing the cells in amaturation factor, such as forskolin or other compound that elevatesintracellular cAMP levels, such as cholera toxin,isobutylmethylxanthine, dibutyladenosine cyclic monophosphate, or otherfactors such as c-kit ligand, retinoic acid, or neurotrophins.Particularly effective are neurotrophin-3 (NT-3) and brain-derivedneurotrophic factor (BDNF). Other candidates are GDNF, BMP-2, and BMP-4.Alternatively or in addition, maturation can be enhanced by withdrawingsome or all of the factors that promote neural precursor proliferation,such as EGF or FGF.

[0096] For use in therapeutic and other applications, it is oftendesirable that populations of precursor or mature neurological cells besubstantially free of undifferentiated pPS cells. One way of depletingundifferentiated stem cells from the population is to transfect themwith a vector in which an effector gene under control of a promoter thatcauses preferential expression in undifferentiated cells. Suitablepromoters include the TERT promoter and the OCT-4 promoter. The effectorgene may be directly lytic to the cell (encoding, for example, a toxinor a mediator of apoptosis). Alternatively, the effector gene may renderthe cell susceptible to toxic effects of an external agent, such as anantibody or a prodrug. Exemplary is a herpes simplex thymidine kinase(tk) gene, which causes cells in which it is expressed to be susceptibleto ganciclovir. Suitable pTERT-tk constructs are provided inInternational Patent Publication WO 98/14593 (Morin et al.).

[0097] Characteristics of neural precursors and terminallydifferentiated cells

[0098] Cells can be characterized according to a number of phenotypiccriteria. The criteria include but are not limited to microscopicobservation of morphological features, detection or quantitation ofexpressed cell markers, enzymatic activity, or neurotransmitters andtheir receptors, and electrophysiological function.

[0099] Certain cells embodied in this invention have morphologicalfeatures characteristic of neuronal cells or glial cells. The featuresare readily appreciated by those skilled in evaluating the presence ofsuch cells. For example, characteristic of neurons are small cellbodies, and multiple processes reminiscent of axons and dendrites. Cellsof this invention can also be characterized according to whether theyexpress phenotypic markers characteristic of neural cells of variouskinds.

[0100] Markers of interest include but are not limited to β-tubulin III,microtubule-associated protein 2 (MAP-2), or neurofilament,characteristic of neurons; glial fibrillary acidic protein (GFAP),present in astrocytes; galactocerebroside (GaIC) or myelin basic protein(MBP), characteristic of oligodendrocytes; Oct-4, characteristic ofundifferentiated hES cells; Nestin, characteristic of neural precursorsand other cells; and both A2B5 and polysialylated NCAM, as alreadydescribed. While A2B5 and NCAM are instructive markers when studyingneural lineage cells, it should be appreciated that these markers cansometimes be displayed on other cell types, such as liver or musclecells. β-Tubulin III was previously thought to be specific for neuralcells, but it has been discovered that a subpopulation of hES cells isalso β-tubulin III positive. MAP-2 is a more stringent marker for fullydifferentiated neurons of various types.

[0101] Tissue-specific markers listed in this disclosure and known inthe art can be detected using any suitable immunological technique—suchas flow immunocytochemistry for cell-surface markers,immunohistochemistry (for example, of fixed cells or tissue sections)for intracellular or cell-surface markers, Western blot analysis ofcellular extracts, and enzyme-linked immunoassay, for cellular extractsor products secreted into the medium. Expression of an antigen by a cellis said to be “antibody-detectable” if a significantly detectable amountof antibody will bind to the antigen in a standard immunocytochemistryor flow cytometry assay, optionally after fixation of the cells, andoptionally using a labeled secondary antibody or other conjugate (suchas a biotin-avidin conjugate) to amplify labeling.

[0102] The expression of tissue-specific gene products can also bedetected at the mRNA level by Northern blot analysis, dot-blothybridization analysis, or by reverse transcriptase initiated polymerasechain reaction (RT-PCR) using sequence-specific primers in standardamplification methods. See U.S. Pat. No. 5,843,780 for further details.Sequence data for the particular markers listed in this disclosure canbe obtained from public databases such as GenBank (URLwww.ncbi.nlm.nih.gov: 80/entrez). Expression at the mRNA level is saidto be “detectable” according to one of the assays described in thisdisclosure if the performance of the assay on cell samples according tostandard procedures in a typical controlled experiment results inclearly discernable hybridization or amplification product. Expressionof tissue-specific markers as detected at the protein or mRNA level isconsidered positive if the level is at least 2-fold, and preferably morethan 10- or 50-fold above that of a control cell, such as anundifferentiated pPS cell, a fibroblast, or other unrelated cell type.

[0103] Also characteristic of neural cells, particularly terminallydifferentiated cells, are receptors and enzymes involved in thebiosynthesis, release, and reuptake of neurotransmitters, and ionchannels involved in the depolarization and repolarization events thatrelate to synaptic transmission. Evidence of synapse formation can beobtained by staining for synaptophysin. Evidence for receptivity tocertain neurotransmitters can be obtained by detecting receptors forγ-amino butyric acid (GABA), glutamate, dopamine,3,4-dihydroxyphenylalanine (DOPA), noradrenaline, acetylcholine, andserotonin.

[0104] Differentiation of particular neural precursor cell populationsof this invention (for example, using NT-3 and BDNF) can generate cellpopulations that are at least 20%, 30%, or 40% MAP-2 positive. Asubstantial proportion, say 5%, 10%, 25%, or more of the NCAM or MAP-2positive cells will be capable of synthesizing a neurotransmitter, suchas acetylcholine, glycine, glutamate, norepinephrine, serotonin, orGABA.

[0105] Certain populations of the invention contain NCAM or MAP-2positive cells that are 0.1%, and possibly 1%, 3%, or 5% or more (on acell count basis) that are positive for tyrosine hydroxylase (TH),measured by immunocytochemistry or mRNA expression. This generallyconsidered in the art to be a marker for dopamine synthesizing cells.

[0106] To elucidate further mature neurons present in a differentiatedpopulation, the cells can be tested according to functional criteria.For example, calcium flux can be measured by any standard technique, inresponse to a neurotransmitter, or other environmental condition knownto affect neurons in vivo. First, neuron-like cells in the populationare identified by morphological criteria, or by a marker such as NCAM.The neurotransmitter or condition is then applied to the cell, and theresponse is monitored (Example 6). The cells can also be subjected tostandard patch-clamp techniques, to determine whether there is evidencefor an action potential, and what the lag time is between appliedpotential and response. Differentiation of neural precursor populationsof this invention can generate cultures that contain subpopulations thathave morphological characteristics of neurons, are NCAM or MAP-2positive, and show responses with the following frequency: a response toGABA, acetylcholine, ATP, and high sodium concentration in at leastabout 40%, 60% or 80% of the cells; a response to glutamate, glycine,ascorbic acid, dopamine, or norepinephrine in at least about 5%, 10% or20% of the cells. A substantial proportion of the NCAM or MAP-2 positivecells (at least about 25%, 50%, or 75%) can also show evidence of anaction potential in a patch-clamp system.

[0107] Other desirable features consistent with functioning neurons,oligodendrocytes, astrocytes, and their precursors can also be performedaccording to standard methods to confirm the quality of a cellpopulation according to this invention, and optimize conditions forproliferation and differentiation of the cells.

[0108] Telomerization of neural precursors It is desirable that neuralprecursors have the ability to replicate in certain drug screening andtherapeutic applications, and to provide a reservoir for the generationof differentiated neuronal and glial cells. The cells of this inventioncan optionally be telomerized to increase their replication potential,either before or after they progress to restricted developmental lineagecells or terminally differentiated cells. pPS cells that are telomerizedmay be taken down the differentiation pathway described earlier; ordifferentiated cells can be telomerized directly.

[0109] Cells are telomerized by genetically altering them bytransfection or transduction with a suitable vector, homologousrecombination, or other appropriate technique, so that they express thetelomerase catalytic component (TERT), typically under a heterologouspromoter that increases telomerase expression beyond what occurs underthe endogenous promoter. Particularly suitable is the catalyticcomponent of human telomerase (hTERT), provided in International PatentApplication WO 98/14592. For certain applications, species homologs likemouse TERT (WO 99/27113) can also be used. Transfection and expressionof telomerase in human cells is described in Bodnar et al., Science279:349, 1998 and Jiang et al., Nat. Genet. 21:111, 1999. In anotherexample, hTERT clones (WO 98/14592) are used as a source of hTERTencoding sequence, and spliced into an EcoRI site of a PBBS212 vectorunder control of the MPSV promoter, or into the EcoRi site ofcommercially available pBABE retrovirus vector, under control of the LTRpromoter.

[0110] Differentiated or undifferentiated pPS cells are geneticallyaltered using vector containing supernatants over a 8-16 h period, andthen exchanged into growth medium for 1-2 days. Genetically alteredcells are selected using 0.5-2.5 μg/mL puromycin, and recultured. Theycan then be assessed for hTERT expression by RT-PCR, telomerase activity(TRAP assay), immunocytochemical staining for hTERT, or replicativecapacity. The following assay kits are available commercially forresearch purposes: TRAPeze® XL Telomerase Detection Kit (Cat. s7707;Intergen Co., Purchase N.Y.); and TeloTAGGG Telomerase PCR ELISApIus(Cat. 2,013,89; Roche Diagnostics, Indianapolis Ind.). TERT expressioncan also be evaluated at the mRNA by RT-PCR. Available commercially forresearch purposes is the LightCycler TeloTAGGG hTERT quantification kit(Cat. 3,012,344; Roche Diagnostics). Continuously replicating colonieswill be enriched by further culturing under conditions that supportproliferation, and cells with desirable phenotypes can optionally becloned by limiting dilution.

[0111] In certain embodiments of this invention, pPS cells aredifferentiated into multipotent or committed neural precursors, and thengenetically altered to express TERT. In other embodiments of thisinvention, pPS cells are genetically altered to express TERT, and thendifferentiated into neural precursors or terminally differentiatedcells. Successful modification to increase TERT expression can bedetermined by TRAP assay, or by determining whether the replicativecapacity of the cells has improved.

[0112] Other methods of immortalizing cells are also contemplated, suchas transforming the cells with DNA encoding myc, the SV40 large Tantigen, or MOT-2 (U.S. Pat. No. 5,869,243, International PatentApplications WO 97/32972 and WO 01/23555). Transfection with oncogenesor oncovirus products is less suitable when the cells are to be used fortherapeutic purposes. Telomerized cells are of particular interest inapplications of this invention where it is advantageous to have cellsthat can proliferate and maintain their karyotype—for example, inpharmaceutical screening, and in therapeutic protocols wheredifferentiated cells are administered to an individual in order toaugment CNS function.

[0113] Use of Neural Precursors and Terminally Differentiated Cells

[0114] This invention provides a method to produce large numbers ofneural precursor cells and mature neuronal and glial cells. These cellpopulations can be used for a number of important research, development,and commercial purposes.

[0115] The cells of this invention can be used to prepare a cDNA libraryrelatively uncontaminated with cDNA preferentially expressed in cellsfrom other lineages. For example, multipotent neural progenitor cellsare collected by centrifugation at 1000 rpm for 5 min, and then mRNA isprepared from the pellet by standard techniques (Sambrook et al.,supra). After reverse transcribing into cDNA, the preparation can besubtracted with cDNA from any or all of the following cell types: cellscommitted to the neuronal or glial cell lineage, mature neurons,astrocytes, oligodendrocytes, or other cells of undesired specificity.This produces a select cDNA library, reflecting transcripts that arepreferentially expressed in neuronal precursors compared with terminallydifferentiated cells. In a similar fashion, cDNA libraries can be madethat represent transcripts preferentially expressed in neuronal or glialprecursors, or mature neurons, astrocytes, and oligodendrocytes.

[0116] The differentiated cells of this invention can also be used toprepare antibodies that are specific for markers of multipotent neuralprogenitors, cells committed to the neuronal or glial cell lineage, andmature neurons, astrocytes, and oligodendrocytes. This inventionprovides an improved way of raising such antibodies because cellpopulations are enriched for particular cell types compared with pPScell cultures, and neuronal or glial cell cultures extracted directlyfrom CNS tissue.

[0117] Polyclonal antibodies can be prepared by injecting a vertebrateanimal with cells of this invention in an immunogenic form. Productionof monoclonal antibodies is described in such standard references asHarrow & Lane (1988), U.S. Pat. Nos. 4,491,632, 4,472,500 and 4,444,887,and Methods in Enzymology 73B: 3 (1981). Other methods of obtainingspecific antibody molecules (optimally in the form of single-chainvariable regions) involve contacting a library of immunocompetent cellsor viral particles with the target antigen, and growing out positivelyselected clones. See Marks et al., New Eng. J. Med. 335:730, 1996,International Patent Applications WO 94/13804, WO 92/01047, WO 90/02809,and McGuiness et al., Nature BiotechnoL 14:1449, 1996. By positivelyselecting using pPS of this invention, and negatively selecting usingcells bearing more broadly distributed antigens (such as differentiatedembryonic cells) or adult-derived stem cells, the desired specificitycan be obtained. The antibodies in turn can be used to identify orrescue neural cells of a desired phenotype from a mixed cell population,for purposes such as costaining during immunodiagnosis using tissuesamples, and isolating precursor cells from terminally differentiatedneurons, glial cells, and cells of other lineages.

[0118] Gene Expression Analysis

[0119] The cells of this invention are also of interest in identifyingexpression patterns of transcripts and newly synthesized proteins thatare characteristic for neural precursor cells, and may assist indirecting the differentiation pathway or facilitating interactionbetween cells. Expression patterns of the differentiated cells areobtained and compared with control cell lines, such as undifferentiatedpPS cells, other types of committed precursor cells (such as pPS cellsdifferentiated towards other lineages, cells committed to the neuronalor glial cell lineage), other types of putative neural stem cells suchas those obtained from neural crest, neurospheres, or spinal chord, orterminally differentiated cells, such as mature neurons, astrocytes,oligodendrocytes, smooth muscle cells, and Schwann cells.

[0120] Suitable methods for comparing expression at the protein levelinclude the immunoassay or immunohistochemistry techniques describedabove. Suitable methods for comparing expression at the level oftranscription include methods of differential display of mRNA (Liang,Peng, et al., Cancer Res. 52:6966, 1992), whole-scale sequencing of cDNAlibraries, and matrix array expression systems.

[0121] The use of microarray in analyzing gene expression is reviewedgenerally by Fritz et al Science 288:316, 2000; “ Microarray BiochipTechnology”, L Shi, www.Gene-Chips.com. Systems and reagents forperforming microarray analysis are available commercially from companiessuch as Affymetrix, Inc., Santa Clara CA; Gene Logic Inc., Columbia MD;HySeq Inc., Sunnyvale CA; Molecular Dynamics Inc., Sunnyvale CA;Nanogen, San Diego CA; and Synteni Inc., Fremont CA (acquired by IncyteGenomics, Palo Alto CA).

[0122] Solid-phase arrays are manufactured by attaching the probe atspecific sites either by synthesizing the probe at the desired position,or by presynthesizing the probe fragment and then attaching it to thesolid support (U.S. Pat. Nos. 5,474,895 and 5,514,785). The probingassay is typically conducted by contacting the array by a fluidpotentially containing the nucleotide sequences of interest undersuitable conditions for hybridization conditions, and then determiningany hybrid formed.

[0123] An exemplary method is conducted using a Genetic Microsystemsarray generator, and an Axon GenePixTM Scanner. Microarrays are preparedby first amplifying cDNA fragments encoding marker sequences to beanalyzed, and spotted directly onto glass slides To compare mRNApreparations from two cells of interest, one preparation is convertedinto Cy3-labeled cDNA, while the other is converted into Cy5-labeledcDNA. The two cDNA preparations are hybridized simultaneously to themicroarray slide, and then washed to eliminate non- specific binding. heslide is then scanned at wavelengths appropriate for each of the labels,the resulting fluorescence is quantified, and the results are formattedto give an indication of the relative abundance of mRNA for each markeron the array.

[0124] Identifying expression products for use in characterizing andaffecting differentiated cells of this invention involves analyzing theexpression level of RNA, protein, or other gene product in a first celltype, such as a pluripotent neuronal precursor cell of this invention,or a cell capable of differentiating along the neuronal or glialpathway; then analyzing the expression level of the same product in acontrol cell type; comparing the relative expression level between thetwo cell types, (typically normalized by total protein or RNA in thesample, or in comparison with another gene product expected to beexpressed at a similar level in both cell types, such as a house-keepinggene); and then identifying products of interest based on thecomparative expression level.

[0125] Drug Screening

[0126] Neural precursor cells of this invention can be used to screenfor factors (such as solvents, small molecule drugs, peptides,polynucleotides) or environmental conditions (such as culture conditionsor manipulation) that affect the characteristics of neural precursorcells and their various progeny.

[0127] In some applications, pPS cells (undifferentiated ordifferentiated) are used to screen factors that promote maturation intoneural cells, or promote proliferation and maintenance of such cells inlong-term culture. For example, candidate maturation factors or growthfactors are tested by adding them to cells in different wells, and thendetermining any phenotypic change that results, according to desirablecriteria for further culture and use of the cells.

[0128] Other screening applications of this invention relate to thetesting of pharmaceutical compounds for their effect on neural tissue ornerve transmission. Screening may be done either because the compound isdesigned to have a pharmacological effect on neural cells, or because acompound designed to have effects elsewhere may have unintended sideeffects on the nervous system. The screening can be conducted using anyof the neural precursor cells or terminally differentiated cells of theinvention, such as dopaminergic, serotonergic, cholinergic, sensory, andmotor neurons, oligodendrocytes, and astrocytes.

[0129] The reader is referred generally to the standard textbook “Invitro Methods in Pharmaceutical Research”, Academic Press, 1997, andU.S. Pat. No. 5,030,015. Assessment of the activity of candidatepharmaceutical compounds generally involves combining the differentiatedcells of this invention with the candidate compound, either alone or incombination with other drugs. The investigator determines any change inthe morphology, marker phenotype, or functional activity of the cellsthat is attributable to the compound (compared with untreated cells orcells treated with an inert compound), and then correlates the effect ofthe compound with the observed change.

[0130] Cytotoxicity can be determined in the first instance by theeffect on cell viability, survival, morphology, and the expression ofcertain markers and receptors. Effects of a drug on chromosomal DNA canbe determined by measuring DNA synthesis or repair. [³H]-thymidine orBrdU incorporation, especially at unscheduled times in the cell cycle,or above the level required for cell replication, is consistent with adrug effect. Unwanted effects can also include unusual rates of sisterchromatid exchange, determined by metaphase spread. The reader isreferred to A. Vickers (pp 375-410 in “In vitro Methods inPharmaceutical Research,” Academic Press, 1997) for further elaboration.

[0131] Effect of cell function can be assessed using any standard assayto observe phenotype or activity of neural cells, such as receptorbinding, neurotransmitter synthesis, release or uptake,electrophysiology, and the growing of neuronal processes or myelinsheaths—either in cell culture or in an appropriate model.

[0132] Therapeutic Use

[0133] This invention also provides for the use of neural precursorcells to restore a degree of central nervous system (CNS) function to asubject needing such therapy, perhaps due to an inborn error infunction, the effect of a disease condition, or the result of an injury.

[0134] To determine the suitability of neural precursor cells fortherapeutic administration, the cells can first be tested in a suitableanimal model. At one level, cells are assessed for their ability tosurvive and maintain their phenotype in vivo. Neural precursor cells areadministered to immunodeficient animals (such as nude mice, or animalsrendered immunodeficient chemically or by irradiation) at an observablesite, such as in the cerebral cavity or in the spinal chord. Tissues areharvested after a period of a few days to several weeks or more, andassessed as to whether pPS derived cells are still present.

[0135] This can be performed by administering cells that express adetectable label (such as green fluorescent protein, orβ-galactosidase); that have been prelabeled (for example, with BrdU or[3H]thymidine), or by subsequent detection of a constitutive cell marker(for example, using human-specific antibody). Where neural precursorcells are being tested in a rodent model, the presence and phenotype ofthe administered cells can be assessed by immunohistochemistry or ELISAusing human-specific antibody, or by RT-PCR analysis using primers andhybridization conditions that cause amplification to be specific forhuman polynucleotide sequences. Suitable markers for assessing geneexpression at the mRNA or protein level are provided elsewhere in thisdisclosure.

[0136] Various animal models for testing restoration of nervous systemfunction are described in “CNS Regeneration: Basic Science and ClinicalAdvances”, M. H. Tuszynski & J. H. Kordower, eds., Academic Press, 1999.

[0137] Differentiated cells of this invention can also be used fortissue reconstitution or regeneration in a human patient in needthereof. The cells are administered in a manner that permits them tograft or migrate to the intended tissue site and reconstitute orregenerate the functionally deficient area.

[0138] Certain neural progenitor cells embodied in this invention aredesigned for treatment of acute or chronic damage to the nervous system.For example, excitotoxicity has been implicated in a variety ofconditions including epilepsy, stroke, ischemia, Huntington's disease,Parkinson's disease and Alzheimer's disease. Certain differentiatedcells of this invention may also be appropriate for treatingdysmyelinating disorders, such as Pelizaeus-Merzbacher disease, multiplesclerosis, leukodystrophies, neuritis and neuropathies. Appropriate forthese purposes are cell cultures enriched in oligodendrocytes oroligodendrocyte precursors to promote remyelination.

[0139] By way of illustration, neural stem cells are transplanteddirectly into parenchymal or intrathecal sites of the central nervoussystem, according to the disease being treated. Grafts are done usingsingle cell suspension or small aggregates at a density of25,000-500,000 cells per μL (U.S. Pat. No. 5,968,829). The efficacy oftransplants of motor neurons or their precursors can be assessed in arat model for acutely injured spinal cord as described by McDonald et al. (Nat. Med. 5:1410, 1999). A successful transplant will showtransplant-derived cells present in the lesion 2-5 weeks later,differentiated into astrocytes, oligodendrocytes, and/or neurons, andmigrating along the cord from the lesioned end, and an improvement ingate, coordination, and weight-bearing.

[0140] The neural progenitor cells and terminally differentiated cellsaccording to this invention can be supplied in the form of apharmaceutical composition, comprising an isotonic excipient preparedunder sufficiently sterile conditions for human administration. Forgeneral principles in medicinal formulation, the reader is referred toCell Therapy. Stem Cell Transplantation, Gene Therapy, and CellularImmunotherapy, by G. Morstyn & W. Sheridan eds, Cambridge UniversityPress, 1996; and Hematopoletic Stem Cell Therapy, E. D. Ball, J. Lister& P. Law, Churchill Livingstone, 2000.

[0141] The composition may optionally be packaged in a suitablecontainer with written instructions for a desired purpose, such as thereconstitution of CNS function to improve some neurological abnormality.

The Following Examples are Provided as Further Non-limitingIllustrations of Particular Embodiments of the Invention. EXAMPLES

[0142] Experimental Procedures

[0143] This section provides details of some of the techniques andreagents used in the Examples below.

[0144] hES cells are maintained either on primary mouse embryonicfibroblasts, or in a feeder-free system. The hES cells are seeded assmall clusters either on irradiated mouse embryonic fibroblasts, or onplates coated with Matrigel® (1:10 to 1:30 in culture medium). hES cellcultures on feeder cells are maintained in a medium composed of 80% KODMEM (Gibco) and 20% Serum Replacement (Gibco), supplemented with 1%non-essential amino acids, 1 mM glutamine, 0.1 mM β-mercaptoethanol and4 ng/mL human bFGF (Gibco). Cultures free of feeder cells are maintainedin the same medium that has previously been conditioned by culturingwith embryonic fibroblasts, and resupplemented with 4 ng/mL bFGF(replaced daily).

[0145] Cells are expanded by serial passaging. The monolayer culture ofES colonies is treated with 1 mg/mL collagenase for 5-20 minutes at 37°C. The cultures are then gently scraped to remove the cells. Theclusters are gently dissociated, and replated as small clusters ontofresh feeder cells.

[0146] Embryoid bodies are produced as follows. Confluent monolayercultures of hES cells are harvested by incubating in 1 mg/mL collagenasefor 5-20 min, following which the cells are scraped from the plate. Thecells are then dissociated into clusters and plated in non-adherent cellculture plates (Costar) in a medium composed of 80% KO (“knockout”) DMEM(Gibco) and 20% non-heat-inactivated FBS (Hyclone), supplemented with 1%non-essential amino acids, 1 mM glutamine, 0.1 mM β-mercaptoethanol. Thecells are seeded at a 1:1 or 1:2 ratio in 2 mL medium per well (6 wellplate). The EBs are fed every other day by the addition of 2 mL ofmedium per well.

[0147] When the volume of medium exceeds 4 mL/well, the EBs arecollected and resuspended in fresh medium. After 4-8 days in suspension,the EBs are plated onto a substrate and allowed to differentiatefurther, in the presence of selected differentiation factors.

[0148] Differentiating into neural precursors is typically performed onwells coated with fibronectin (Sigma) at a final concentration of 20μg/mL in PBS. Using 1 mL/well (9.6 cm ²), plates are incubated at 4° C.overnight or at room temperature for 4 h. The fibronectin is thenremoved, and the plates are washed with PBS or KO DMEM once before use.

[0149] Immunocytochemistry for NCAM and A2B5 expression is conducted asfollows: Live cells are incubated in primary antibody diluted in culturemedium with 1% goat serum for 15 minutes at 37° C., washed once withmedium, and then incubated with labeled secondary antibody for 15 min.After washing, the cells are then fixed for 15-20 min in 2%paraformaldehyde. For other markers, cultures are fixed for 10-20 minwith 4% paraformaldehyde in PBS, washed 3 times with PBS, permeabilizedfor 2 min in 100% ethanol, and washed with 0.1 M PBS. Cultures are thenincubated in a blocking solution of 0.1 M PBS with 5% NGS (normal goatserum) for at least 1 hour at room temperature. Cultures are thenincubated in primary antibody diluted in 0.1M PBS containing 1% NGS forat least 2 h at room temperature. They are then washed in PBS before a30 min incubation with secondary antibody in the same buffer. Antibodiesused include those shown in Table 1. TABLE 1 Antibody for Neural CellPhenotypic Markers Anti- Working Epitope body Isotype DilutionSpecificity Source 5A5 mouse IgM 1:1 Polysialylat- Developmental ed NCAMstudies hybridoma bank A2B5 mouse IgM 1:1 ganglioside ATCC-CRL1520 clone105 β-tub- IgG 1:1000 Sigma T-8660 ulin GFAP rabbit polyclonal 1:500DAKO 2-334 IgG GalC mouse IgG3 1:25 Boehringer 1351- 621

[0150] Bead immunosorting is conducted using the following reagents andequipment: magnetic cell separator; Midi MACs™ column; buffer of PBS CMFcontaining 0.5% BSA and 2 mM EDTA; primary antibody against NCAM orA2B5; rat anti-mouse IgG (or IgM) microbeads; pre-separation filter; ratanti-mouse kappa PE; and a FACScan device. Cells are harvested usingtrypsinlEDTA (Gibco) and dissociated. After removing the trypsin, thecells are resuspended in MACs™ buffer. Cells are then labeled withprimary antibody for 6-8 min at room temp., and washed 2 times in MACs™buffer by spinning cells at 300×g for 10 min and aspirating the buffer.The cells are then resuspended in 80 μI (minimum vol.) per 10⁷ cells. 20μI (minimum vol.) MACs ram™ IgG microbeads per 10⁷ cells are added for15 min at 6-12° C. The sample is then washed 2 times in MACs™ bufferbefore magnetic separation. With the column in the magnetic cellseparator, the cell suspension is applied to the column (LS+Midi) in˜3-5 mL buffer. Negative cells are passed through by washing 3 timeswith 3 mL of MACs™ buffer. The column is then removed from the magneticfield, and positive cells are eluted with 5 mL of MACs™ buffer.

[0151] After separation, A2B5+ or NCAM+ cells are maintained on platescoated with poly-lysine and laminin in DMEM/F12 (Biowhittaker)supplemented with N2 (Gibco 17502-014), B27 (Gibco 17504-010) and thefactors indicated. Source of the factors is shown in Table 2. TABLE 2Factors used for Neural Cell Culture Working Growth Factor SourceConcentration human EGF R & D Systems 10 ng/mL human bFGF Gibco 10-25ng/mL human CNTF R & D Systems 1-10 ng/mL human PDGF R & D Systems 1ng/mL human IGF-I R & D Systems 1 ng/mL

[0152] RT-PCR analysis of expression at the transcription level isconducted as follows: RNA is extracted from the cells using RNAeasy Kit™(Qiagen) as per manufacturer's instructions. The final product is thendigested with DNAse to get rid of contaminating genomic DNA. The RNA isincubated in RNA guard (Pharmacia Upjohn) and DNAse I (Pharmacia Upjohn)in buffer containing 10 mM Tris ph 7.5, 10 mM MgCI₂, and 5 mM DDT at37°C. for 30-45 min. To remove protein from the sample, phenolchloroform extraction is performed, and the RNA precipitated with 3 Msodium acetate and 100% cold ethanol. The RNA is washed with 70%ethanol, and the pellet is air-dried and resuspended in DEPC-treatedwater.

[0153] For the reverse transcriptase (RT) reaction, 500 ng of total RNAis combined with a final concentration of 1×First Strand Buffer (Gibco),20 mM DDT and 25,μg/mL random hexamers (Pharmacia Upjohn). The RNA isdenatured for 10 min at 70° C., followed by annealing at roomtemperature for 10 min. dNTPs are added at a final concentration of 1 mMalong with 0.5μL of Superscript II RT (Gibco), incubated at 42 ° C. for50 minutes, and then heat-inactivated at 80 ° C. for 10 min. Samples arethen stored at −20 ° C. till they are processed for PCR analysis.Standard polymerase chain reaction (PCR) is performed using primersspecific for the markers of interest in the following reaction mixture:cDNA 1.0,μL, 10×PCR buffer (Gibco) 2.5 μL, 10×MgCI₂ 2.5 μL, 2.5 mM dNTP3.0 μL, 5 μM 3′-primer 1.0 μL, 5 μM 5′-primer, 1.0 μL, Taq 0.4,μL,DEPC-water 13.6 μL.

Example 1 NCAM-Positive Cells

[0154] This experiment focused on determining whether the humanembryonic stem cells (hES) could undergo directed differentiation toNCAM-positive progenitor cells. The hES cells were harvested either frommEF-supported cultures or feeder-free cultures, and then differentiatedvia embryoid body (EB) formation in suspension culture using mediumcontaining 20% FBS. The EBs were then plated intact onto fibronectin inDMEM/F12 medium, supplemented with N2 supplement (Gibco) and 25 ng/mLhuman bFGF. After culturing for about 2-3 days, NCAM-positive cells andA2B5-positive cells were identified by immunostaining.

[0155] Magnetic bead sorting and immunopanning were both successful inenriching NCAM-positive cells. The starting population of cellstypically contained 25-72% NCAM-positive cells. After immuno-isolation,the NCAM-positive proportion was enriched to 43-72%. Results are shownin Table 3. TABLE 3 Differentiation and Sorting Conditions for NCAMpositive Cells Cells staining positively Factors used in for NCAM hESCell Line used Differentiation Positive Negative for DifferentiationCulture Type of Sort Before sort sort sort H13 p28 CFN bead sort 33 9241 H13p28 CFN panning 25 n/a n/a H9 p32 CFN panning 64 72 51 H1 p32 CFNbead sort 27 77  9 H9 p19 CFN bead sort 58 76 32 H9 p31 545.184 CFN beadsort 50 91 67 H1 p40 545.185 CFN bead sort A 65 89 31 H1 p40 545.185 CFNbead sort B 63 81 33 H7NG p28/4 545.187 CFN bead sort A 53 92 45 H7NGp28/4 545.187 CFN bead sort A 72 87 50 H1p39 545.189 CFNIP bead sort 1643  6 H7 p32 667.004 CFNIP bead sort 25 73 10 H1p43 667.010 CFNIP beadsort 47 86 31 H1p44 667.012 CFNIP bead sort 52 89 34 H1 p46 667.020 EPFIbead sort 60 23  8 H1 p47 667.031 EPFI—EPFI bead sort 53 91 27 H1 p47667.033 CFN-F bead sort 41 76 24 H9 p40MG 667.038 EPFI bead sort 55 8025

[0156] In the first 10 experiments shown, NCAM positive cells retrievedfrom the sort were plated on poly-L-lysine/laminin in DMEM/F12 with N2and B27 supplements and 2 mg/mL BSA, 10 ng/mL human CNTF, 10 ng/mL humanbFGF and 1 ng/mL human NT-3. In subsequent experiments, cells weremaintained in DMEM/F12 with N2 and B27 supplements and 10 ng/mL EGF, 10ng/mL bFGF, 1 ng/mL PDGF, and 1 ng/mL IGF-1.

[0157]FIG. 1 (Upper Panel) shows the growth curves for the NCAM positivecells. The cells studied in this experiment were prepared by formingembryoid bodies in 20% FBS for 4 days in suspension, then plating onto afibronectin matrix in DMEM/F12 with N2 and B27 supplements and 25 ng/mLbFGF for 2-3 days. The cells were then positively sorted for NCAMexpression, and maintained in a medium containing CNTF, bFGF, and NT3.The sorted cells did not show increased survival relative to theunsorted population. It was found that some of the NCAM positive cellsalso express β-tubulin 111, indicating that these cells have thecapacity to form neurons. They also had morphology characteristic ofneuronal cells. There were also A2B5 positive cells within thispopulation, which may represent glial progenitor cells. However, veryfew cells were positive for GFAP, a marker for astrocytes. Although thiscell population proliferated in culture, the proportion of NCAM positivecells (and the capacity to form neurons) diminished after severalpassages.

Example 2 A2B5-Positive Cells

[0158] Cells in this experiment were immunoselected for the surfacemarker A2B5. hES cells were induced to form EBs in 20% FBS. After 4 daysin suspension, the EBs were plated onto fibronectin in DMEM/F12 with N2and B27 supplemented with 10 ng/mL human EGF, 10 ng/mL human bFGF, 1ng/mL human IGF-l, and 1 ng/mL human PDGF-AA. After 2-3 days in theseconditions, 25-66% of the cells express A2B5. This population isenriched by magnetic bead sorting to 48-93% purity (Table 4). TABLE 4Differentiation and Sorting Conditions for A2B5-positive Cells Cellsstaining positively Factors used in for NCAM hES Cell Line used forDifferentiation Positive Negative Differentiation Culture Type of SortBefore sort sort sort H7 p32 667.004 CFNIP bead sort 25 77 10 H1p43667.010 CFNIP bead sort 62 n/a 50 H1 p44 667.012 CFNIP bead sort 56 8932 H1 p46 667.020 EPFI bead sort 27 48  2 H1 p47 667.032 EPFI bead sort57 93 30 H9 p40MG 667.038 EPFI bead sort 66 93 41 H9 p42 667.041 EPFIbead sort 27 70  6

[0159]FIG. 2 shows an exemplary procedure for obtaining A2B5-positivecells. Abbreviations used: MEF-CM=medium conditioned by culturing withmouse embryonic fibroblasts; +/−SHH=with or without sonic hedgehog;

[0160] D/F12=DMEM/F12 medium; N2 and B27, culture supplements (Gibco);EPFI=growth factors EGF, PDGF, bFGF, and IGF-1; PLL=poly-L lysinesubstrate; PLL/FN=substrate of poly-L lysine and fibronectin.

[0161]FIG. 1 (Lower Panel) shows the growth curves for the sortedA2B5-positive cells. The cells were maintained in the same mediaformulation on poly-l-lysine coated plates. The cells proliferate whenserially passaged.

Example 3 Maturation of A2B5-Positive Cells

[0162] A2B5-positive cells were induced to differentiate by the additionof forskolin. These cells have been assessed through different culturepassages, as shown in Table 5. TABLE 5 Phenotypic Features of MatureNeural Cells Neuron-like No. of passages Method of morphology CellsStaining Positively for: after A2B5 sort Maturation visible β-tubulinGFAP GalC A2B5 NCAM 1 PICNT + Fk yes 38 ± 9% 13 ± 7% 79 ± 3% 28 ± 6% 4days 3 PICNT + Fk yes +++ + +++++ ++ 2 days 7 +/− EF yes + + ++ +++ −+/− serum

[0163] Even though the cells were sorted for A2B5 expression, thepopulation demonstrated the capacity to generate not onlyoligodendrocytes, and astrocytes, but also a large proportion ofneurons. This is surprising: it was previously thought that A2B5expressing cells were glial precursors, and would give rise tooligodendrocytes, and astrocytes—while NCAM expressing cells wereneuronal precursors, giving rise to mature neurons. This experimentdemonstrates that pPS cells can be differentiated into a cell populationthat proliferates repeatedly in culture, and is capable of generatingneurons and glia.

Example 4 Transplantation of Differentiated Cells into the MammalianBrain

[0164] Transplantation of neural precursor cells was done using cellsderived from two hES cell lines: the line designated H1, and agenetically altered line designated H7NHG. The H7NHG cell line carriesan expression cassette that permits the cells to constitutively expressgreen fluorescent protein (GFP).

[0165] Neonatal Sprague Dawley rats received unilateral intrastriatalimplants of one of the following cell populations:

[0166] undifferentiated hES cells

[0167] embryoid bodies derived from hES cells

[0168] neural precursors sorted for NCAM expression (Example 1)

[0169] neural precursors sorted for A2B5 expression (Example 2)

[0170] Control animals received grafts of irradiated mouse embryonicfibroblasts upon which the undifferentiated hES cells were maintained.To determine if cell proliferation occurred after grafting, some animalswere pulsed with intraperitoneal injections of BrdU, commencing 48 hprior to sacrifice. Fourteen days after transplantation, the rats weretranscardially perfused with 4% paraformaldehyde and the tissue wasprocessed for immunohistochemical analysis.

[0171]FIG. 3 shows the fluorescence observed in sections from animalsadministered cells expressing GFP.

[0172] Surviving cells were detected in all transplanted groups. Theundifferentiated cells presented as large cell masses, suggestingunregulated growth with areas of necrosis and vacuolation of surroundingtissue (Left-side Panels). Immunostaining for AFP in an animaltransplanted with Hi cells showed that undifferentiated hES cellstransformed into visceral endoderm after transplantation. Embryoidbodies remained in the graft core with little migration, and were alsosurrounded by areas of necrosis. (Middle Panels). In contrast, sortedNCAM-positive cells appeared as single cells and showed some degree ofmigration distal to the site of implantation.

Example 5 Differentiation to Mature Neurons

[0173] To generate terminally differentiated neurons, the first stage ofdifferentiation was induced by forming embryoid bodies in FBS mediumwith or without 10 μM retinoic acid (RA). After 4 days in suspension,embryoid bodies were plated onto fibronectin-coated plates in definedmedium supplemented with 10 ng/mL human EGF, 10 ng/mL human bFGF, 1ng/mL human PDGF-AA, and 1 ng/mL human IGF-1. The embryoid bodiesadhered to the plates, and cells began to migrate onto the plastic,forming a monolayer.

[0174] After 3 days, many cells with neuronal morphology were observed.The neural precursors were identified as cells positive for BrdUincorporation, nestin staining, and the absence of lineage specificdifferentiation markers. Putative neuronal and glial progenitor cellswere identified as positive for polysialylated NCAM and A2B5. Forty oneto sixty percent of the cells expressed NCAM, and 20-66% expressed A2B5,as measured by flow cytometry. A subpopulation of the NCAM-positivecells was found to express β-tubulin III and MAP-2. There was noco-localization with glial markers such as GFAP or GaIC. The A2B5positive cells appeared to generate both neurons and glia. Asubpopulation of the A2B5 cells expressed β-tubulin III or MAP-2, and aseparate subpopulation expressed GFAP. Some of the cells with neuronalmorphology double-stained for both A2B5 and NCAM. Both the NCAM positiveand A2B5 positive populations contained far more neurons than glia.

[0175] The cell populations were further differentiated by replating thecells in a medium containing none of the mitogens, but containing 10ng/mL Neurotrophin-3 (NT-3) and 10 ng/mL brain-derived neurotrophicfactor (BDNF). Neurons with extensive processes were seen after about 7days. Cultures derived from embryoid bodies maintained in retinoic acid(RA) showed more MAP-2 positive cells (˜26%) than those maintainedwithout RA (˜5%). GFAP positive cells were seen in patches. GaICpositive cells were identified, but the cells were large and flat ratherthan having complex processes.

[0176] A summary of cell types and markers expressed at different stagesof differentiation is provided in Table 6. TABLE 6 Phenotypic Markers(Immunocytochemistry) Undifferentiated hES colonies NCAM-positiveprogenitors A2B5 positive progenitors Tra-1-60 + Nestin subset Nestinsubset Tra-1-81 + A2B5 subset NCAM subset SSEA-4 + β-tubulin III subsetβ-tubulin III subset β-tubulin III + + Map-2 subset Map-2 subset Nestin− GFAP − GFAP rare Map-2 − GalC − GalC − Neurofilament (NF) − AFP − AFP− GFAP − muscle-specific actin − muscle-specific actin − GalC −α-fetoprotein − muscle-specific actin − NCAM − A2B5 − Neurons AstrocytesOligodendrocytes β-tubulin III + GFAP + GalC + MAP-2 + Neurofilament(NF) subset GABA subset tyrosine hydroxylase subset glutamate subsetglycine subset

[0177] The presence of neurotransmitters was also assessed.GABA-immunoreactive cells were identified that co-expressed β-tubulinIlIl or MAP2, and had morphology characteristic of neuronal cells.Occasional GABA-positive cells were identified that did not co-expressneuronal markers, but had an astrocyte-like morphology. Neuronal cellswere identified that expressed both tyrosine hydroxylase (TH) and MAP-2.Synapse formation was identified by staining with synaptophysinantibody.

[0178]FIG. 4 shows TH staining in cultures differentiated from the H9line of human ES cells. Embryoid bodies were maintained in 10 μMretinoic acid for 4 days, then plated onto fibronectin coated plates inEGF, basic FGF, PDGF and IGF for 3 days. They were next passaged ontolaminin in N2 medium supplemented with 10 ng/mL NT-3 and 10 ng/mL BDNF,and allowed to differentiate further for 14 days. The differentiatedcells were fixed with 2% formaldehyde for 20 min at room temp, and thendeveloped using antibody to TH, a marker for dopaminergic cells.

Example 6 Calcium Imaging

[0179] Standard fura-2 imaging of calcium flux was used to investigatethe functional properties of the hES cell derived neurons.Neurotransmitters studied included GABA, glutamate (E), glycine (G),elevated potassium (50 mM K⁺instead of 5 mM K⁺), ascorbic acid(control), dopamine, acetylcholine (ACh) and norepinephrine. Thesolutions contained 0.5 mM of the neurotransmitter (except ATP at 10 pM)in rat Ringers (RR) solution: 140 mM NaCI, 3 mM KCI, 1 mM MgCI₂, 2 mMCaCI₂, 10 mM HEPES buffer, and 10 mM glucose. External solutions wereset to pH 7.4 using NaOH. Cells were perfused in the recording chamberat 1.2-1.8 mL/min, and solutions were applied by bath application usinga 0.2 mL loop injector located ˜0.2 mL upstream of the bath import.Transient rises in calcium were considered to be a response if thecalcium levels rose above 10% of the baseline value within 60 sec ofapplication, and returned to baseline within 1-2 min.

[0180]FIG. 5 shows the response of neural-restricted precursors tovarious neurotransmitters. Panel A shows the ratio of emission data fromsingle cells on two different coverslips. Addition of theneurotransmitters is indicated above by labeled triangles.

[0181] Panel B shows the frequency of cells tested that responded tospecific neurotransmitters. Panel C shows the combinations ofneurotransmitter responses observed. Of the 53 cells tested, 26responded to GABA, acetylcholine, ATP and elevated potassium. Smallersubsets of the population responded to other combinations of agonists.Only 2 of the cells failed to respond to any of the agonists applied.

Example 7 Electrophysiology

[0182] Standard whole-cell patch-clamp technique was conducted on thehES cell derived neurons, to record ionic currents generated involtage-clamp mode and the action potential generated in current-clampmode. The external bath solution was rat Ringers solution (Example 6).The internal solution was 75 mM potassium-aspartate, 50 mM KF, 15 mMNaCI, 11 mM EGTA, and 10 mM HEPES buffer, set to pH 7.2 using KOH.

[0183] All 6 cells tested expressed sodium and potassium currents, andfired action potentials. Passive membrane properties were determinedwith voltage steps from −70 to −80 mV; and produced the following data:average capacitance (C_(m))=8.97±1.17 pF; membrane resistance(R_(m))=487.8±42.0 MΩ; access resistance (R_(a))=23.4±3.62 MΩ. Ioniccurrents were determined by holding the cells at −100 mV, and steppingto test voltages between −80 and 80 mV in 10 mV increments, producingthe following data: average sodium current I_(Na)=−531.8±136.4 pA;average potassium current I_(K=)441.7±113.1 pA;I_(Na)(density)=−57.7±7.78 pA/pF; I_(K)(density)=48.2±10.4 pA/pF.

[0184]FIG. 6 shows results from a typical experiment. Panel A showssodium and potassium currents observed in two cells depolarized to testpotentials between −80 and 80 mV from a holding potential of −100 mV.Panel B shows the inward (Na⁺) and outward (K⁺) peak current-voltagerelationships observed. Sodium current activates between −30 and 0 mV,reaching a peak at −10 or 0 mV. Potassium current activates above −10mV, becoming equal or larger in magnitude than the sodium current atvoltages between 20 and 40 mV. Panel C shows action potentials generatedby the same cells n response to depolarizing stimuli. Cell membraneswere held at voltages between −60 and −100 mV in −80 or −150 pA ofcurrent, and depolarized for short durations

Example 8 Dopaminergic Cells Derived From Neural Progenitor Ells

[0185] Embryoid bodies were cultured in suspension with 10 μM retinoicacid for 4 days, then plated into defined medium supplemented with EGF,bFGF, PDGF, and IGF-1 for 3-4 days. Cells were then separated bymagnetic bead sorting or immunopanning into A2B5-positive orNCAM-positive enriched populations.

[0186] The immuno-selected cells were maintained in defined mediumsupplemented with 10 ng/mL NT-3 and 10 ng/mL BDNF. After 14 days, 25±4%of the NCAM-sorted cells were MAP-2 positive—of which 1.9±0.8% wereGABA-positive, and 3 ±1% were positive for tyrosine hydroxylase (TH):the rate-limiting enzyme for dopamine synthesis, generally considered tobe representative of dopamine-synthesizing cells.

[0187] In the cell population sorted for NCAM, the cells that wereNCAM+ve did not express glial markers, such as GFAP or GaIC. These dataindicate that a population comprising neuron restricted precursors canbe isolated directly from hES cell cultures, essentially uncontaminatedwith glial precursors.

[0188] Cells sorted for A2B5, on the other hand, have the capacity togenerate both neurons and astrocytes. After the enrichment, the cellswere placed into defined media supplemented with NT-3 and BDNF andallowed to differentiate for 14 days. Within the first 1-2 days afterplating, cells in the A2B5 enriched population began to extendprocesses. After two weeks, cells took on the morphology of matureneurons, and 32±3% of the cells were MAP-2 positive. Importantly, 3±1%of the MAP-2 cells were TH-positive, while only 0.6±0.3% were GABAimmunoreactive. These data indicate that a population of cells can beobtained from hES cells that comprise progenitors for both astrocytesand neurons, including those that synthesize dopamine.

[0189] Further elaboration of conditions for obtaining TH-expressionneurons was conduced as follows. Embryoid bodies were generated fromconfluent hES cells of the H7 line at passage 32 by incubating in 1mg/mL collagenase (37° C., 5-20 min), scraping the dish, and placing thecells into non-adherent culture plates (Costar®). The resulting EBs werecultured in suspension in media containing FBS and 10 μM all-transretinoic acid. After four days, the aggregates were collected andallowed to settle in a centrifuge tube. The supernatant was thenaspirated, and the aggregates were plated onto poly L-lysine andfibronectin coated plates in proliferation medium (DMEM/F12 1:1supplemented with N2, half-strength B27, 10 ng/mL EGF (R&D Systems), 10ng/mL bFGF (Gibco), 1 ng/mL PDGF-AAA (R&D Systems), and 1 ng/mL IGF-1 (R& D Systems).

[0190] The EBs were allowed to attach and proliferate for three days;then collected by trypsinizing ˜1 min (Sigma) and plated at 1.5×10⁵cells/well onto poly l-lysine and laminin coated 4-well chamber slidesin proliferation medium for one day. The medium was then changed toNeural Basal medium supplemented with B27, and one of the followinggrowth cocktails:

[0191] 10 ng/mL bFGF (Gibco), 10 ng/mL BDNF, and 10 ng/mL NT-3

[0192] 10 ng/mL bFGF, 5000 ng/mL sonic hedgehog, and 100 ng/mL FGF8b

[0193] 10 ng/mL bFGF alone

[0194] The cells were maintained in these conditions for 6 days, withfeeding every other day. On day 7, the medium was changed to NeuralBasal medium with B27, supplemented with one of the following cocktails:

[0195] 10 ng/mL BDNF, 10 ng/mL NT-3

[0196] 1 μM cAMP, 200 μM ascorbic acid

[0197] 1 μM cAMP, 200 μM ascorbic acid, 10 ng/mL BDNF, 10 ng/mL NT-3

[0198] The cultures were fed every other day until day 12 when they werefixed and labeled with anti-TH or MAP-2 for immunocytochemistry.Expression of the markers was quantified by counting four fields in eachof three wells using a 40X objective lens.

[0199] Results are shown in Table 7. Initial culturing in bFGF, BDNF andNT-3 yielded the highest proportion of TH positive cells. TABLE 7Conditions for Producing Dopaminergic Neurons Culture conditions % MAP-2% MAP-2 cells that are days 1-6 days 6-12 positive TH positive B, N, FB, N 26% 5.5% B, N, F CA, AA 35% 4.0% B, N, F CA, AA, B, N 25% 8.7% F,F8, S B, N 37% 3.7% F, F8, S CA, AA 34% 3.9% F, F8, S CA, AA, B, N 21%5.8% F B, N 28% 3.5% F CA, AA 26% 4.1% F CA, AA, B, N 22% 5.7%

[0200] It is understood that certain adaptations of the inventiondescribed in this disclosure are a matter of routine optimization forthose skilled in the art, and can be implemented without departing fromthe spirit of the invention, or the scope of the appended claims.

What is claimed as the invention is:
 1. A cell population thatproliferates in an in vitro culture, obtained by differentiating primatepluripotent stem (pPS) cells, wherein at least ˜30% of the cells in thepopulation are committed to form neuronal cells, glial cells, or both.2. A cell population that proliferates in an in vitro culture, obtainedby differentiating primate pluripotent stem (pPS) cells, comprising atleast ˜60% neural progenitor cells, wherein at least 10% of the cellscan differentiate into neuronal cells, and at least 10% of the cells candifferentiate into glial cells.
 3. A cell population that proliferatesin an in vitro culture, obtained by differentiating primate pluripotentstem (pPS) cells, comprising at least ˜60% neural progenitor cells,wherein at least 10% of the cells express A2B5, and at least 10% of thecells express NCAM.
 4. The cell population of claim 1, wherein the pPScells are human embryonic stem (hES) cells.
 5. The cell population ofclaim 1, obtained by differentiating pPS cells in a medium containing atleast two ligands that bind growth factor receptors, selected from thegroup consisting of EGF, bFGF, PDGF, IGF-1, and antibodies to receptorsfor these ligands.
 6. The cell population of claim 1, obtained bydifferentiating pPS cells in a medium containing growth factors, sortingthe differentiated cells for expression of NCAM or A2B5, and thencollecting the sorted cells.
 7. The cell population of claim 1, whichcan be induced to produce a population of cells of which at least 30% ofthe cells have morphological features of mature neurons and are NCAMpositive.
 8. The cell population of claim 7, wherein the cells havingmorphological features of mature neurons have at least three of thefollowing characteristics: a) at least 60% of the cells show calciumflux when administered acetylcholine; b) at least 60% of the cells showcalcium flux when administered GABA; c) at least 10% of the cells showcalcium flux when administered norepinephrine; d) at least 60% of thecells show calcium flux when subjected to an external potassiumconcentration of 50 mM; or e) at least 25% of the cells demonstrateaction potentials when subject to stimulation in a whole-cell patchclamp apparatus.
 9. The cell population of claim 1, which can be inducedto produce a population of cells in which at least 1% of the cells stainpositively for tyrosine hydroxylase.
 10. The cell population of claim 1,comprising cells genetically altered to express telomerase reversetranscriptase.
 11. A cell population comprising mature neurons,astrocytes, oligodendrocytes, or any combination thereof, obtained byfurther differentiating the cell population according to claim
 1. 12.The cell population of claim 11, comprising a subpopulation of at least30% of the cells that have the morphological characteristics of neuronsand are NCAM positive, wherein the subpopulation has the followingproperties: a) at least 60% show calcium flux when administeredacetylcholine; b) at least 60% show calcium flux when administered GABA;c) at least 10% show calcium flux when administered norepinephrine; d)at least 60% show calcium flux when subjected to an external potassiumconcentration of 50 mM; or e) at least 25% demonstrate action potentialswhen subject to stimulation in a whole-cell patch clamp apparatus. 13.The cell population of claim 11, in which at least 1% of the cells stainpositively for tyrosine hydroxylase.
 14. The cell population of claim11, obtained by culturing the cell population of claim 1 in a mediumcontaining an activator of cAMP, a neurotrophic factor, or a combinationthereof.
 15. An isolated neural precursor cell, obtained by providingthe cell population of claim 1, and selecting therefrom a cell havingcharacteristics of a neural precursor cell.
 16. An isolated matureneuron, astrocyte, or oligodendrocyte, obtained by providing the cellpopulation of claim 11, and selecting therefrom a cell havingcharacteristics of a neuron, astrocyte, or oligodendrocyte,respectively.
 17. The isolated mature neuron of claim 16, which is adopaminergic neuron.
 18. The isolated mature neuron of claim 16,obtained by culturing the cell of claim 1 in a medium containing anactivator of cAMP, a neurotrophic factor (such as nerve growth factor,neurotrophin 3, or brain-derived neurotrophic factor), or a combinationthereof.
 19. A cell population comprising at least ˜60% neuralprogenitor cells and/or mature neurons that have the same genome as anestablished human embryonic stem (hES) cell line.
 20. The cellpopulation of claim 18, wherein the neural progenitor cells and/ormature neurons express NCAM, A2B5, MAP-2, or Nestin.
 21. A method forobtaining neural precursor cells capable of producing a cell populationcomprising at least 1% tyrosine hydroxylase positive cells, comprisingdifferentiating human embryonic stem cells.
 22. The method of claim 21,wherein the differentiating comprises culturing in a medium containingat least two ligands that bind growth factor receptors, selected fromthe group consisting of EGF, bFGF, PDGF, IGF-1, and antibodies toreceptors for these ligands.
 23. A method for obtaining a cellpopulation comprising at least 1% tyrosine hydroxylase positive cells,comprising differentiating human embryonic stem cells.
 24. The method ofclaim 21, comprising obtaining neural precursor cells according to claim19, and then culturing the cells obtained thereby in a medium containingan activator of cAMP, a neurotrophic factor (such as nerve growthfactor, neurotrophin 3, or brain-derived neurotrophic factor), or acombination thereof.
 25. The method of claim 21, further comprisinggenetically altering the cells to express telomerase reversetranscriptase before or after differentiating.
 26. A method of screeninga compound for neural cell toxicity or modulation, comprising combiningthe compound with a cell population according to claim 1; determiningany phenotypic or metabolic changes in the cell that result from contactwith the compound; and correlating the change with neural cell toxicityor modulation.
 27. A method of screening a compound for neural celltoxicity or modulation, comprising combining the compound with a cellpopulation according to claim 11; determining any phenotypic ormetabolic changes in the cell that result from contact with thecompound; and correlating the change with neural cell toxicity ormodulation.
 28. A method for obtaining a polynucleotide comprising anucleotide sequence contained in an mRNA more highly expressed in neuralprogenitor cells, the method comprising: a) determining the level ofexpression of a plurality of mRNAs in one or more cells in the cellpopulation of claim 1, in comparison to the level of expression of thesame mRNAs in more mature neural cells; b) identifying an mRNA expressedat a higher level in the cell(s) from the cell population, relative tothat in the more mature cells; and c) preparing a polynucleotidecomprising a nucleotide sequence of at least 30 consecutive nucleotidescontained in the mRNA selected in step b).
 29. A method ofreconstituting or supplementing central nervous system (CNS) function inan individual, comprising administering to the individual a cellpopulation according to claim
 1. 30. A method of reconstituting orsupplementing CNS function in an individual, comprising administering tothe individual a cell population according to claim 11.